Project Summary/Abstract Nucleotide-based second messengers play important biological functions in living organisms. Among various second messenger molecules, several of them are generated by families of NTases that belong to Polb superfamily. In animals, cytosolic NTases OAS and cGAS generate 2?-5?-linked oligoadenylates and 2?- 5?-linked c-GAMP, respectively, both of which trigger innate immune response against viral and bacterial infection. In bacteria, NTase DncV generates 3?-5?-linked cGAMP, which activates CapV to inhibit cell growth. Using a combination of comparative genomics, sequence conservation, and structure analysis, Aravind and coworkers uncovered a vast network of nucleotide-centric systems in bacteria. A family of NTases named SMODS (secondary messenger oligo and dinucleotides synthase) was predicted to generate second messengers. And several conserved domains were predicted to be receptors of the second messengers generated by SMODS. Among the predicted receptors, the domain named SAVED (SMODS-associated and fused to various effector domains) is the most abundant. With the exception of a recent characterization of the product generated by one of SMODS, however, the predicted biochemical and biological functions of SMODS and SAVED have not been experimentally tested. Employing approaches of bioinformatics, biochemistry, and structural biology, we aim to pursue the following two lines of investigation of SMODS and SAVED: 1) We will in vitro reconstitute the enzymatic activity of SMODS and probe interaction between SAVED and the second messengers generated by SMODS; and 2) We will carry out structural studies of SMODS and SAVED, SMODS in complex with its activator and substrates, and SAVED in complex with the second messengers generated by SMODS.